Studies on the regulation of cell growth and division are central to understanding human cell proliferative diseases such as cancer. One approach to identify these important regulatory factors is to first understand the mechanism of DNA replication in eukaryotic cells and identify the replication proteins that may be regulated. In turn, these proteins can be used as substrates to identify the key regulators. To this end, we have been studying DNA replication from the Simian Virus 40 (SV40) origin using a cell-free replication system. The DNA tumor virus, SV40 has long been a model for understanding macromolecular processes such as DNA replication and transcription and for studies on cell transformation by oncogenes. A cell free system for the replication of DNA containing the SV40 origin of DNA replication has been extensively characterized and a number of replication factors have been purified from human cells. These in turn have been used to characterize the mechanism of DNA replication in eukaryotes. One such protein, called replication factor A, is overproduced in human tumor and transformed cells. This protein functions in the initiation of DNA replication, but also stimulates the activity of both DNA polymerases alpha and delta, during the elongation stage of DNA replication. We have shown that RF-A is phosphorylated in a cell cycle dependent manner in both human and yeast cells. The goal of this proposed research is to understand the regulation of DNA replication in eukaryotic cells, using the SV40 DNA replication system as the foundation for the proposed research. In particular, the cell cycle dependent phosphorylation of replication factor A will be further characterized, particularly the identification of the cell cycle regulated protein kinase that phosphorylates it in S phase. How phosphorylation affects the function of the protein will also be investigated. Finally, the distribution of sites of DNA replication and RF-A in the nucleus will be studied to determine if temporal regulation of these structures occurs.